Because periodontal ligament (PDL) cells are reported to contain progenitor or stem cell populations, they are considered a beneficial cell source for clinical periodontal regeneration. Both bone morphogenetic protein 4 (BMP4) and human telomerase reverse transcriptase (hTERT) have essential roles in the modulation of stem cell properties. In this study we report for the first time that the combined ectopic expression of BMP4 and hTERT significantly enhanced the multipotent differentiation efficiency and capacity of human PDL fibroblasts (PFs), as shown by osteogenic, adipogenic and neurogenic differentiation in vitro, and cementum/PDL-like tissue regeneration in vivo. These findings may be attributed, at least in part, to the original upregulation of important stem cell markers, such as scleraxis, Stro-1 and CD146, and the extremely lowered threshold for BMP concentration to activate BMP signaling by enhanced basal phosphorylation levels of Smad 1/5/8. In addition, the significantly reduced expression levels of CD146 and CD90 with the presence of Noggin confirms the direct effect of BMP4 on the stem cell-like phenotype of genetically modified PF cells (BT-PFs). Furthermore, BT-PFs exhibited a high neural differentiation capacity (>75%). After transplantation into NOD/SCID mice, genetically modified-PFs generated cementum/PDL-like structures on the surface of the carrier. The multipotency of these modified cells potentially provides an attractive source of stem cells for therapeutic purposes and regenerative medicine.